This invention relates generally to ligand-receptor interactions and more specifically to growth differentiation factor receptor proteins and the ligands that bind to such receptors and methods of use therefor.
The transforming growth factor xcex2 (TGF-xcex2) superfamily encompasses a group of structurally-related proteins which affect a wide range of differentiation processes during embryonic development. The family includes, Mullerian inhibiting substance (MIS), which is required for normal male sex development (Behringer, et al., Nature, 345:167, 1990), Drosophila decapentaplegic (DPP) gene product, which is required for dorsal-ventral axis formation and morphogenesis of the imaginal disks (Padgett, et al., Nature, 325:81-84, 1987), the Xenopus Vg-1 gene product, which localizes to the vegetal pole of eggs ((Weeks, et al., Cell, 51:861-867, 1987), the activins (Mason, et al., Biochem, Biophys. Res. Commun., 135:957-964, 1986), which can induce the formation of mesoderm and anterior structures in Xenopus embryos (Thomsen, et al., Cell, 63:485, 1990), and the bone morphogenetic proteins (BMPS, osteogenin, OP-1) which can induce de novo cartilage and bone formation (Sampath, et al., J. Biol. Chem., 265:13198, 1990). The TGF-xcex2s can influence a variety of differentiation processes, including adipogenesis, myogenesis, chondrogenesis, hematopolesis, and epithelial cell differentiation (for review, see Massague, Cell 49:437, 1987).
The proteins of the TGF-xcex2 family are initially synthesized as a large precursor protein which subsequently undergoes proteolytic cleavage at a cluster of basic residues approximately 110-140 amino acids from the C-terminus. The C-terminal regions, or mature regions, of the proteins are all structurally related and the different family members can be classified into distinct subgroups based on the extent of their homology. Although the homologies within particular subgroups range from 70% to 90% amino acid sequence identity, the homologies between subgroups are significantly lower, generally ranging from only 20% to 50%. In each case, the active species appears to be a disulfide-linked dimer of C-terminal fragments. Studies have shown that when the pro-region of a member of the TGF-xcex2 family is coexpressed with a mature region of another member of the TGF-xcex2 family, intracellular dimerization and secretion of biologically active homodimers occur (Gray, A. et al., Science, 247:1328, 1990). Additional studies by Hammonds, et al., (Molec. Endocrin. 5:149, 1991) showed that the use of the BMP-2 pro-region combined with the BMP-4 mature region led to dramatically improved expression of mature BMP-4. For most of the family members that have been studied, the homodimeric species has been found to be biologically active, but for other family members, like the inhibins (Ling, et al., Nature, 321 :779, 1986) and the TGF-xcex2s (Cheifetz, et al., Cell, 48:409, 1987), heterodimers have also been detected, and these appear to have different biological properties than the respective homodimers.
The study of receptor-ligand interactions has revealed a great deal of information about how cells respond to external stimuli. This knowledge has led to the development of therapeutically important compounds, such as erythropoietin, colony stimulating factors and PDGF.
The present invention provides receptors for the growth differentiation factor (GDF) growth factor family. These receptors are useful for identifying antagonists and agonists for agricultural and human therapeutic purposes.
In a first embodiment, the invention provides a recombinant cell line that expresses growth differentiation factor-8 (GDF-8) or growth differentiation factor-11 (GDF-11) receptor polypeptide. Also included are antibodies that bind to GDF receptors, polynucleotides encoding the receptors and the GDF receptor proteins themselves.
Peptide fragments of GDF receptors, such as the GDF-8 or GDF-11 receptors, are also included. Such peptides may be useful in inhibiting binding of GDF-8 or GDF-11 to either its own receptor or another GDF-receptor (e.g., GDF-8 and -11 may bind the same receptor).
In another embodiment, the invention provides a substantially purified GDF-8-binding agent, wherein the binding agent inhibits GDF-8 binding to GDF-8 receptor. Such agents that inhibit GDF-11 binding are also included.
In yet another embodiment, the invention provides a method for identifying a GDF receptor polypeptide including incubating components such as GDF polypeptide and a cell expressing a receptor or a soluble receptor under conditions sufficient to allow the GDF to bind to the receptor; measuring the binding of the GDF polypeptide to the receptor; and isolating the receptor.
The invention also includes a method for identifying a compound that binds to GDF receptor polypeptide including incubating components comprising the compound and GDF polypeptide under conditions sufficient to allow the components to interact and measuring the binding or effect of binding of the compound to GDF receptor polypeptide.
The invention also provides non-human transgenic animals that have a phenotype characterized by expression of GDF-receptor polypeptide, the phenotype being conferred by a transgene contained in the somatic and germ cells of the animal, the transgene comprising a nucleic acid sequence which encodes GDF-receptor polypeptide. Methods of producing such transgenic animals are also included.
In another embodiment, the invention includes a method for inhibiting the expression of GDF-receptor in a cell including contacting GDF-receptor with an inhibiting effective amount of an antisense oligonucleotide that binds to a segment of an mRNA transcribed from a GDF-receptor gene, whereby the binding of the antisense to the mRNA segment inhibits GDF-receptor expression.